EP0877754B1 - Amino acid-enriched plant protein reserves, particularly lysine-enriched maize gamma-zein, and plants expressing such proteins - Google Patents

Abstract

An oligonucleotide including at least one concatenation coding for a polypeptide of formula (P-K)<SUB>n</SUB>, wherein n is an integer of at least 2, P is a proline amino acid residue, K is a lysine amino acid residue, and the sign "-" is a bond, particularly a peptide bond, between the two amino acid residues. The n (P-K) units are also bound together by such bonds, e.g. peptide bonds.

Description

The present application relates to new means for the preparation of plants expressing deficient amino acid-enriched reserve proteins in normal reserve proteins, and in particular lysine enriched reserve proteins. The subject of the invention is also the reserve proteins thus modified, as well as plants expressing these modified reserve proteins. Among these reserve proteins, the invention relates to zein family reserve proteins.

Many plants, if necessary after transformation by physico-chemical steps, are of major economic importance in human or animal nutrition, and the problem of improving their nutritional quality has already given rise to different types of research. In particular, to remedy the insufficient presence of certain amino acids in plant reserve proteins, selected varieties with superior nutritional qualities have been developed, or proposed various modifications using the use of engineering techniques. genetics in order to favor or increase the production in these plants, in particular certain deficit amino acids, which are nevertheless important for the nutritional qualities of the plant. These deficient amino acids are, for example, lysine, methionine.

In the context of the present application, the inventors have proposed to provide an original solution to the problem of improving plants, especially the improvement of their nutritional qualities, and for this purpose worked primarily from a plant of considerable economic importance, corn. Specifically, they focused on the endosperm reserve proteins of maize seeds, proteins which comprise in particular zeins and, in particular, γ-zein.

During the development of corn seeds, the endosperm cells synthesize large amounts of reserve proteins, including α-, β-, and γ-zeines. These zeins are accumulated in the protein bodies derived from the endoplasmic reticulum (ER).

In general, zeins represent a complex group of proteins, divided into several groups, α-, β-, γ-, and δ-zeins (Larkins et al, 1989), encoded by a multigene family (Hagen and Rubenstein). 1980, Gene 13: 239-249). Although they have a variable structure, these proteins share common characteristics: the presence in their primary structure of tandem repeats rich in Proline-type amino acid residues, the presence of numerous hydrophobic residues which confer their insolubility on these proteins. in an aqueous medium, and the absence of lysine residues, essential amino acids for humans and monogastric animals. The absence of lysine in all the major proteins (detected in large quantities in the endosperm) naturally produced from the zein family leads to an unbalanced amino acid composition in maize seeds.

Among these proteins, maize γ-zein is a protein having a molecular weight of 28 kDa, the coding sequence of which has been described in the form of cDNA by Prat et al (Nucleic Acids Research, Vol.13, No. 5, 1985, pp. 1494-1504). The complete sequence of the gene encoding γ-zein, including the upstream and downstream non-coding sequences containing the expression regulatory elements, has been described by Reina, M. et al (Nucleic Acids Research, vol. 18, No. 21, 1990, p 6426).

So far, different approaches have been considered to increase the lysine content of proteins of the zein family. In this respect, genetic and molecular approaches have been implemented. For example, mutants allowing the production of lysine-rich maize, such as the opaque-2 (o2) mutant and the floury-2 (fl-2) mutant (Mertz et al., 1964, Science 145, 279-280). Nelson et al., 1965, Science 150, 1469-1470) have thus been proposed and attempts to overcome the deleterious effects of the absence of certain classes of zeins, in particular α-zeines, on phenotypic characteristics, by corn selection. containing o2 modifier genes (Paez et al., 1969, Plant Sci., 9, 251-252, Geetha et al, 1991, Plant Cell 3.1207-1219) were made.

Another approach has been to perform an indirect action on free lysine production, especially in dicotyledonous plants. This technique has been implemented by acting on the deregulation of the key enzymes (DHTPS and AK) involved in the cycle of lysine biosynthesis via aspartate. A significant increase in free lysine levels was obtained in the leaves, but not in the seeds, in tobacco plant transformation experiments with E. coli bacteria containing the dapA genes and E. coli bacteria containing the lysc gene. (Shaul and Galili, 1992, Plant J. 2, 203-209 and 1993, Plant Mol Biol 23, 759-768, Perl, A., Schaul O., Galili, G., 1992, Plant Molecular Biology, 19. , pp. 815-823). Recently, the same Corynebacterium dapA and E. coli lysC genes have been used and expressed under the control of a seed-specific promoter in soybean plants. Expression of these two enzymes in soybean resulted in a five-fold increase in seed lysine content (Falco et al., 1995, BIO-Technology 13, 577-582).

Other authors (Wallace et al., 1988, Science 240, 662-664) have attempted to increase the lysine in the α-zein (19 kDa) corn seed by spot incorporation of lysine residues at different positions. in the α-zein molecule. The expression of these constructs in Xenopus oocytes led to the correct assembly of lysine-rich zeins in the vesicles analogous to the protein bodies. However, normal α-zein and the modified lysine-enriched form were degraded when expressed in tobacco seeds (Othani et al., 1991, Plant Mol Biol 16: 117).

So far, there was no knowledge of the means by which the expression of a lysine-enriched zein in cells producing it naturally in corn, ie in endosperm cells, could be achieved. A fortiori, the expression in other plant cells of zeins enriched in lysine was not controlled.

One of the aims of the invention is therefore to propose means for obtaining a lysine-enriched zein, in particular a lysine-enriched maize γ-zein, this protein being expressed in particular in the corn seed cells and in particular in the endosperm cells, said modified protein being further expressed such that its localization and accumulation properties in the endoplasmic reticulum and derived protein bodies are preserved.

The expression "lysine-enriched" means, in the context of the present application, that the protein has an increased number of lysine residues relative to the natural protein from which it is derived, for example as a result of a modification of the sequence of nucleotides that expresses it.

The invention also proposes means for obtaining the expression of proteins, preferably lysine-enriched γ-zeins, in plant cells of different tissues, for example leaf or root tissues, and this, where appropriate, in tissues. plant cells of plants which do not naturally express the protein, in particular the γ-zein whose production is desired.

Moreover, according to a particular embodiment of the invention, other reserve proteins can be enriched in lysine in analogous ways, and more particularly reserve proteins of the family of zeins.

The inventors initially proposed, in order to carry out the invention, to introduce into the gene coding for γ-zein or for other reserve proteins maize or other plants, or in the coding sequence of this gene, sequences coding for lysine-enriched polypeptides, in order to obtain the production of γ-zeins or other proteins enriched in lysine and, therefore, seeds enriched in lysine. Different sites of the coding sequence of the γ-zein gene have been identified as permissive sites, (also called "neutral sites") to prepare nucleotide sequences thus modified.

The present application therefore proposes means for transforming the gene coding for maize γ-zein or for transforming any nucleotide sequence coding for γ-zein and derived from this gene, so as to obtain from the expression of the gene modified or more generally the modified nucleotide sequence, a protein enriched in lysine; these means comprise in particular synthetic oligonucleotides coding for an amino acid sequence comprising lysine residues.

The subject of the invention is also recombinant nucleotide sequences or chimeric sequences capable of coding for a lysine-enriched γ-zein.

Also within the scope of the invention are host cells transformed with such sequences, and in particular plant cells, for example cells allowing the regeneration of plants, as well as plants or parts of plants (tissues, organs ,. ..) containing such cells and stably producing modified reserve proteins, and in particular lysine-enriched γ-zeines.

The subject of the invention is also said modified proteins, for example enriched in lysine, as well as antibodies directed against these proteins, and more particularly lysine enriched zeol family reserve proteins.

• n est un entier supérieur ou égal à 2,
• P représente un résidu d'acide aminé Proline,
• K représente un résidu d'acide aminé Lysine,
• le signe "-" symbolise une liaison entre les deux résidus d'acides aminés et notamment une liaison de type peptidique, les n unités (P-K) étant liées entre elles également par de telles liaisons par exemple de type peptidique.

An oligonucleotide suitable for carrying out the invention, usable for the preparation of recombinant nucleotide sequences, is characterized in that it comprises at least one sequence coding for a polypeptide corresponding to the formula (PK) _n , in which:

• n is an integer greater than or equal to 2,
• P represents a Proline amino acid residue,
• K represents an amino acid residue Lysine,
• the sign "-" symbolizes a bond between the two amino acid residues and in particular a peptide-type binding, the n units (PK) being linked to each other also by such bonds, for example of the peptide type.

An oligonucleotide according to the invention is therefore characterized according to a first embodiment, in that it comprises a sequence coding for a sequence of repeated units comprising two amino acids.

The codons of the oligonucleotide may be identical for all Proline residues and / or for all lysine residues. They can also be different for the same amino acid residue, the variation taking into account the degeneracy of the genetic code.

Preferably, this oligonucleotide is formed by a coding sequence for more than 2 units (P-K). Preferably n is less than or equal to 30, in particular less than 20 and advantageously n is equal to 4, 5, 6, 7, 8, 9 or 10 or 15.

The "oligonucleotides" according to the invention can be synthesized chemically by any available technique.

The term "polypeptide" with reference to the sequence (PK) _n denotes, in the context of the present application, an acid sequence amino acids with more than 2 amino acid residues and up to 60 amino acid residues.

According to a first variant embodiment of the invention, the oligonucleotide comprises several sequences coding for a polypeptide corresponding to the formula (PK) _n , which are identical or different, associated in tandem.

These oligonucleotides are either repetitions of the same sequence or different linking associations. The number of associated concatenations is variable, for example being between 2 and 10 concatenations.

According to another embodiment of the invention, the oligonucleotide previously defined is characterized in that it comprises at least one sequence coding for a polypeptide corresponding to the formula (PK) _n , in which the sequence of n units (PK ) is interrupted by one or more amino acid residues different from residues P or K.

Preferably, the additional amino acids incorporated in the sequence formed by the units (PK) are chosen so as not to modify the organization of the polypeptide encoded by the oligonucleotide, or at least not to cause interaction with acids. amino acid of a protein in which said polypeptide is incorporated, under conditions that would affect the structure and / or the function and / or the location of this protein.

This can in particular be the case when the number of units (PK) is large or when several sequences formed of sequences coding for units (PK) _n are associated in tandem and that the preparation of the corresponding oligonucleotide requires that are synthesized several nucleotide sequences which are then associated for example by means of linker sequences.

According to another embodiment of the invention, the oligonucleotide is such that the sequence coding for the polypeptide comprising the n units (PK) is completed at its 5 'end and / or at its 3' end, by one or several codons, coding for example for at least one lysine residue at the N-terminus of the polypeptide.

By way of example, a preferred oligonucleotide according to the invention is characterized in that it encodes a polypeptide corresponding to the formula (PK) _n , to the formula K- (PK) ₄ , or to the formula 2K (PK ) ₄ .

According to a particular embodiment, the composition of this oligonucleotide corresponds to one of the sequences described in the following pages and identified by the names SEQ ID No. 1 and SEQ ID No. 2.

The previously described oligonucleotides are basic means for making recombinant nucleotide sequences capable of expressing lysine-enriched plant reserve proteins or polypeptides.

• l'expression de la séquence de nucléotides dans une cellule végétale déterminée permet d'obtenir une protéine de réserve modifiée localisée de façon identique ou similaire dans cette cellule à la protéine de réserve normale qui serait exprimée dans la même cellule, dans les mêmes conditions par l'enchaînement de nucléotides normal codant correspondant, et/ou
• la protéine de réserve modifiée codée par la séquence de nucléotides est reconnue immunologiquement par des anticorps produits contre la protéine de réserve normale correspondante

The subject of the invention is therefore also a recombinant nucleotide sequence comprising a sequence of nucleotides coding for a plant reserve protein, characterized in that it furthermore comprises an oligonucleotide according to the invention, inserted in a site of the sequence of nucleotides chosen in such a way that:

• the expression of the nucleotide sequence in a given plant cell makes it possible to obtain a modified reserve protein identical or similarly localized in this cell to the normal reserve protein which would be expressed in the same cell, under the same conditions by the sequence of corresponding normal nucleotide encoding, and / or
• the modified reserve protein encoded by the nucleotide sequence is immunologically recognized by antibodies produced against the corresponding normal reserve protein

In particular, the aforementioned antibodies are constituted by a polyclonal serum or are obtained against epitopes of the normal reserve protein which are conserved in the modified reserve protein.

The plant cells referred to above include any plant cell, regardless of its tissue origin or nature. In particular, we are interested in the context of the invention to the cells of the reserve organs, but also to the cells of leaves, stems, tubers, etc.

By the term "reserve protein" of a plant is meant in the context of the present application a protein synthesized during the seed maturation and which is used during the germination phase as a main nutrition reserve.

In general, it is a polypeptide capable of being synthesized in a reserve tissue whatever the location in the plant; the reserve proteins used in the context of the present application are in particular those produced in the seeds or seeds of plants of the cereal, cruciferous or leguminous family, and are, for example, prolamins or zeins.

The choice of the insertion site (s) of the oligonucleotide in the sequence coding for the plant reserve protein is determined by the respect of the conditions stated above. Depending on the case, the insertion may take place in a repetitive domain (in terms of amino acid sequence) of the protein or at a C- or N-terminus.

The condition given above according to which the expression of the recombinant nucleotide sequence of the invention in a plant cell makes it possible to obtain a modified reserve protein located identically or similarly to the normal reserve protein which would be expressed in the same conditions in the same plant cell, for example for synthesized γ-zeines, the possibility of being accumulated in the endoplasmic reticulum of plant cells which express it, and in particular in the protein bodies formed from this endoplasmic reticulum, when the protein is expressed in the endosperm cells.

To obtain this result by means of the recombinant nucleotide sequences of the invention, expression systems adapted to the cellular host in which the selected nucleotide sequence is expressed, and in particular the regulatory elements, for example the promoters, are chosen for their functional character in the tissue containing the transformed cells. Tests to make this choice can be made based on the various constructions described in the examples.

In order to verify that the immunological properties of the modified reserve protein expressed by the nucleotide sequence of the invention have not been substantially modified, antisera such as αG2 antiserum described specifically in US Pat. experimental part that follows.

According to a first embodiment of the invention, the recombinant nucleotide sequence is characterized in that it is obtained from a coding sequence of nucleotides which leads to the expression of a naturally lysine-poor reserve protein. .

In general, this recombinant nucleotide sequence codes for a modified reserve protein derived from a reserve protein naturally produced by a plant that can be used for animal or human nutrition.

Thus, the reserve proteins whose lysine content is modified in the context of the present invention are advantageously reserve proteins of plants of the family of cereals, legumes or crucifers. Particularly important reserve proteins are maize, especially zeins, and more especially γ-zein corn, which is sought to increase the lysine content.

A particular recombinant nucleotide sequence according to the invention is characterized in that the coding sequence of nucleotides coding for the maize γ-zein that it contains, corresponds to the sequence presented in FIG. 9.

Other recombinant nucleotide sequences of the invention are characterized in that the encoding sequence of nucleotides they comprise, codes for a reserve protein of a plant chosen from the following: soybean, sunflower, tobacco , wheat, oats, alfalfa, rice, rapeseed, sorghum, Arabidopsis.

According to a preferred embodiment of the invention, in the recombinant nucleotide sequence comprising a sequence coding for the maize γ-zein, the oligonucleotide of the invention is inserted in place of the sequence coding for the Pro domain. -X naturally present in the amino acid sequence of the γ-zein corn or as a result of this sequence. The Pro-X domain of the amino acid sequence of the maize γ-zein consists of the amino acids located between positions 70 and 91 of the amino acid sequence shown in FIG. 9, corresponding to nucleotides 265 to 330 of the sequence shown in Figure 9.

Preferably, in the nucleotide sequence of the invention, the oligonucleotide instead of or following the Pro-X domain present between nucleotides 276 and 357 of the sequence shown in FIG. 9.

According to another embodiment of the invention, in the recombinant nucleotide sequence comprising a sequence coding for the γ-maize zein, the oligonucleotide of the invention is inserted following the Pro-X domain conserved in the sequence maize γ-zein.

According to another embodiment variant, in the recombinant nucleotide sequence comprising a sequence coding for the γ-zein of maize, the oligonucleotide of the invention is inserted into the Pro-X domain maintained in the sequence of the γ-zein.

The insertions referred to above are feasible by the available techniques, for example by recombination of sequences having undergone one or more enzymatic digestion steps.

In a particular embodiment of the invention, a selected reserve protein enriched in a specific amino acid is expressed in heterologous plant cells. In other words, a reserve protein naturally present in a given plant, is expressed in amino acid-enriched form, in another plant, or in a cell other than that which naturally produces it.

In addition to the sequence coding for a plant reserve protein and the oligonucleotide of the invention, the recombinant nucleotide sequences of the invention may also comprise an expression promoter and, for example, a promoter chosen for its specific character. for the expression in certain parts or in certain tissues of the plants, or on the contrary a promoter chosen for its constitutive character. For example, when specific, the promoters may be specific to the seeds and / or specific organs or tissues of plants. They may alternatively or also be specific for a growth phase, for example of a determined stage of germination.

Conversely, the use of constitutive promoters allows the constant and general expression of the reserve protein, resulting in a competition between the expression of the native reserve protein when it is present and the modified reserve protein.

By way of example, advantageous promoters for carrying out the invention are the promoter of the γ-zein of maize contained in the 1.7 kb sequence located upstream of the coding sequence shown in FIG. 7, the cauliflower mosaic virus promoter, namely the CaMV35S promoter (EP-B-0131623), the promoter constituting the gene rice actin 1 (PCT / US 9100073) or the specific high molecular weight gluthenin seed promoter of wheat (Colot V. et al, 1987, EMBO Journal, Vol 6, pp. 3559-3564).

Where appropriate, these promoters are supplemented by other regulatory sequences, and in particular expression enhancers.

By way of example, other promoters that can be used for carrying out the invention are, for example, the promoter of the gene coding for the Arabidopsis thaliana seeds 2S reserve protein, or the promoters of the bean lectin or of the the β-phaseolin of beans.

The additional introduction of expression promoters into the nucleotide sequence control sequences according to the invention also makes it possible to increase the level of the primary transcription of the nucleotide sequence and, where appropriate, to increase the amount of modified reserve proteins produced. Activators are, for example, monocotyledon introns such as intron 1 of the rice actin gene.

The subject of the invention is also a cloning and / or expression vector, characterized in that it comprises, in a non-essential site for its replication, a nucleotide sequence corresponding to one of the previously given definitions. Particular interesting vectors in the context of the embodiment of the invention are, for example, the plasmids pP20γZ, pH30γZ or pH45γZ. The plasmid pP20γZ was deposited at the CNCM (Paris, FRANCE) on October 31, 1995 under the number I-1640. The plasmid pH45γZ was deposited at the CNCM on October 31, 1995 under the number I-1639.

Also within the scope of the invention is a polypeptide as expressed by a recombinant nucleotide sequence as defined above.

The expression "polypeptide" does not introduce, in the context of the present invention, particular limitation as to the number of amino acids forming the polypeptide. It can therefore be sequences comprising a few amino acids, usually designated by the term "peptides", or even much longer sequences such as those of proteins.

In this regard, the invention relates to modified lysine-rich maize γ-zein, characterized in that it is encoded by a recombinant nucleotide sequence described above.

• n est un entier supérieur ou égal à 2,
• P représente un résidu d'acide aminé Proline,
• K représente un résidu d'acide aminé Lysine,
• le signe "-" symbolise une liaison entre les deux résidus d'acides aminés et notamment une liaison de type peptidique, les n unités (P-K) étant liées par des liaisons notamment de type peptidique.

According to a preferred embodiment of the invention, the modified lysine-enriched maize γ-zein is characterized in that its amino acid sequence is modified by at least one polypeptide corresponding to the formula (PK) _n , in which :

• n is an integer greater than or equal to 2,
• P represents a Proline amino acid residue,
• K represents an amino acid residue Lysine,
• the sign "-" symbolizes a bond between the two amino acid residues and in particular a peptide-type bond, the n units (PK) being linked by bonds, in particular of the peptide type.

According to an alternative embodiment of the invention, the polypeptide integrated in the amino acid sequence of γ-zein corresponds to the formula K- (PK) _n .

• lorsque la γ-zéine modifiée riche en lysine est produite dans une cellule hôte, notamment une cellule végétale, elle est localisée de façon identique ou similaire dans cette cellule à la γ-zéine de maïs normale qui serait produite dans les mêmes conditions, dans la même cellule hôte et/ou,
• la γ-zéine de maïs modifiée est reconnue par des anticorps dirigés contre la γ-zéine normale de maïs.

The polypeptides of the invention corresponding to one of the formulas (PK) _n , K- (PK) _n or to variants are substituted for a sequence naturally present in the normal maize γ-zein or inserted with a deletion of a or more amino acids in the amino acid sequence of the normal maize γ-zein, or added in the amino acid sequence of the normal γ-zein, the insertion site of the polypeptide being chosen such that:

• when the modified γ-zein rich in lysine is produced in a host cell, in particular a plant cell, it is located in the same way or similarly in this cell to the normal γ-zein of maize that would be produced under the same conditions, in the same host cell and / or,
• the modified maize γ-zein is recognized by antibodies directed against the normal γ-zein of maize.

The P20γZ proteins shown in FIG. 11 or H30γZ or H45γZ shown in FIG. 10 are preferred examples of the embodiment of the invention and represent modified lysine-enriched maize γ-zeins.

The subject of the invention is also a recombinant host cell, characterized in that it comprises a nucleotide sequence described above.

Suitable host cells are for example bacteria cells, such as those of E. coli or Agrobacterium tumefaciens. Preferably, in the context of the invention, and for the stable expression of the desired modified reserve protein, plant-derived host cells will be used.

For example, these cells of plant origin are seed cells, plant cells, and for example, preferably, corn seed endosperm cells.

The nucleotide sequence of the invention is preferably introduced into the genome of the host cell stably and under conditions such that the expressed protein reserve enriched in acids amino acids and in particular lysine is localized as would be the normal corresponding protein in the same host cell.

Various techniques are available to transform host cells. For example, and in order to stably or transiently transform the host cells, we will use electroporation techniques, bombardment of microprojectiles carrying DNA, via particle gun, via explant culture with Agrobacterium tumefaciens, by penetration of microfibres.

In addition to corn seed endosperm cells, soybean, sunflower, tobacco, wheat, oat, alfalfa, rice, rapeseed, sorghum or Arabidopsis cells can be used to express the nucleotide sequences of the invention.

The present application also relates to seeds producing a polypeptide as described above and plants producing the same polypeptide. Preferably, these plants are corn.

The subject of the invention is also seeds obtained from transformed plants expressing the polypeptide of the invention, in other words the modified reserve polypeptide enriched in determined amino acids.

According to a particularly advantageous embodiment of the invention, modified γ-zein proteins enriched in lysine are expressed in maize opaque 2 mutants. These o2 mutants described by Emerson RA et al. (1935, Cornell Univ., Agric Exp, Stn Mem 180) and characterized by Mertz ET et al (1964, Science 145: 279-280) have their lysine content greatly increased thereby greatly improving the nutritional qualities of corn (compensation its low level in this essential amino acid). Conventional maize has a lysine content of about 0.24% of the raw product (total grain weight), while opaque maize 2 is 0.5% lysine. However, they have insufficient agronomic characteristics because their endosperm is much less vitreous and very friable (starchy phenotype). This has the effect of making them extremely sensitive to pathogenic organisms and post-harvest treatment stages. This phenotype is in fact due to the significant decrease of certain reserve proteins, in particular alpha zeines. In fact Opaque 2 codes for a transcription factor necessary for the expression of certain zein genes (Schmidt RJ et al., 1990, Proc Natl Acad Sci USA 87, 46-50).

Opaque derivatives 2 no longer having the aforementioned drawbacks have been developed by conventional genetic improvement, they are QPM (Quality Protein Maize) maize. Recent genetic analysis of these maize (Lopes M.A. et al., 1995, Theor Appl., Genet 19, 274-281) has shown that only 2 or 3 loci are key in these favorable modifications. Further genetic and biochemical analyzes allow us to postulate that one of the 3 loci responsible is the γ-zein locus: the maize genotypes that carry a duplication of this gene located in the centrometric region of chromosome 7, all turn out to be opaque 2 modifiers (Lopes MA et al, 1995, Mol Gen. Genet 19: 247: 603-613).

The present invention makes it possible to prepare opaque mutant maize 2 from maize having only one γ-zein gene at chromosome 7, which are complemented by the addition of a recombinant sequence coding for a γ-zein. but enriched with lysine. In addition to acquiring hardness properties similar to a non-mutant opaque corn 2, it has the advantage of significantly increasing the lysine content, thus exceeding that of QPM corn.

The present invention makes it possible to obtain modified maize opaque mutants 2 in which a recombinant nucleotide sequence coding for a lysine-enriched maize γ-zein has been inserted.

• a) transformation d'une cellule végétale, avec une séquence de nucléotides ou un vecteur ci-dessus décrits, dans des conditions permettant l'expression de façon stable et fonctionnelle de la protéine de réserve modifiée codée par la séquence de nucléotides;
• b) régénération de plantes à partir de la cellule de plante transformée de l'étape a), pour obtenir des plantes exprimant la protéine de réserve modifiée,
• c) le cas échéant, obtention des semences à partir des plantes modifiées obtenues à l'étape b).

The subject of the invention is also a process for obtaining plants or seeds expressing a modified reserve protein, characterized in that it comprises the steps of:

• a) transforming a plant cell, with a nucleotide sequence or a vector described above, under conditions allowing the stable and functional expression of the modified reserve protein encoded by the nucleotide sequence;
• b) regenerating plants from the transformed plant cell of step a), to obtain plants expressing the modified reserve protein,
• c) where appropriate, obtaining seeds from the modified plants obtained in step b).

According to an advantageous embodiment of the invention, the transformed plant is maize and the enriched modified reserve protein is γ-zein, enriched in lysine.

The invention also relates to the plants obtained by implementing such a method.

In order to evaluate the content of a given amino acid of the plants according to the invention, it is possible to use a dosing protocol such as that mentioned in Zarkadas et al, 1995, J. Agri. Food. Chem. Flight. 43: 84-93.

• Figure 1
  • Carte de restriction du plasmide pP20γZ
• Figure 2
  • Carte de restriction du plasmide pH45γZ
• Figure 3
  • Représentation schématique des protéines codées par les gènes modifiés et non modifiés de la γ-zéine : γ-zéine de type sauvage (γZ), γ-zéines riches en lysine (P20γZ, H30γZ, H45γZ et N13γZ) résultant de l'insertion des oligonucléotides codant pour des séquences riches en lysine. La séquence d'acides aminés des polypeptides insérés est indiquée en utilisant le code à une lettre de la représentation des acides aminés. Les abréviations suivantes sont utilisées :
    • Term : terminal ;
    • ProX DOMAIN : domaine linker proline-Xaa.
• Figure 4 - Analyse in-vitro des γ-zéines riches en lysine. (A) traduction in-vitro et translocation des transcrits correspondant aux γ-zéines modifiées riches en lysine; lignes 1, 5, 9 et 13 : produits complets de traduction ; lignes 2, 6, 10 et 14 : produits complets de traduction après translocation dans les microsomes canins (CM); lignes 3, 7, 11 et 15: produits de translocation résistant à l'action de la protéinase K (PK) ; lignes 4, 8, 12 et 16 : totalité des produits de traduction après traitement avec la protéinase K en présence de 0,5% Nonidet P40(NP40). (B) Immunoprécipitation des produits de traduction in-vitro correspondant aux protéines γ-zéine et γ-zéine modifiées riches en lysine, en utilisant l'anti-sérum αPL. Ligne 1 : γ-zéine ; ligne 2 : P20γZ ; ligne 3 : H30γZ ; ligne 4 : H45γZ et ligne 5 : N13γZ. (C) Même légende que pour (B) mais en utilisant l'antisérum αG2. Les marqueurs de poids moléculaire (en kilodaltons) sont indiqués sur la gauche.
• Figure 5 - Activité tissu-spécifique du promoteur de la γ-zéine. Des endospermes de maïs, des embryons et des feuilles de maïs ont été co-transformés par bombardement de particules en utilisant les constructions représentées dans la figure (dans la partie à droite). Les activités relatives de la luciférase (LUC, colonnes en gris) et de la β-glucuronidase (GUS, colonnes hachurées) ont été exprimées sous la forme d'un multiplicateur des valeurs obtenues avec des projectiles nus ± la déviation standard des différents ratios.
• Figure 6 - Expression des γ-zéines riches en lysine dans l'endosperme sub-aleuronique des cellules. (A) Immunoblot avec un antisérum αPL, de protéines extraites d'endospermes transformés par pN13γZ (ligne 2), pH45γZ (ligne 3), pH30γZ (ligne 4) et pP20γZ (ligne 5). Le témoin (ligne 1) correspond à des endospermes non transformés. Les marqueurs de poids moléculaire (en kilodaltons) sont indiqués à gauche. (B) expression des transcrits H45γZ et N13γZ dans des endospermes transformés de façon transitoire. Les cDNAs obtenus à partir des tissus transformés avec pH45γZ (ligne 2), pN13γZ (ligne 3) et le témoin (ligne 1) ont été amplifiés par PCR et analysés en utilisant un oligonucléotide synthétique codant pour une séquence riche en lysine à titre de sonde.
• Figure 7 - Accumulation de γ-zéines riches en lysine dans les corps protéiques de l'endosperme. (A) Analyse en immunoblot en utilisant l'antisérum αPL, des corps protéiques isolés à partir des endospermes transformés avec pP20γZ (ligne 1), pH30γZ (ligne 2), pH45γZ (ligne 3), pN13γZ (ligne 4) et aucun ADN (ligne 5). (B) Analyse en immunoblot en utilisant l'antisérum αPL, des corps protéiques isolés à partir des endospermes transformés avec pP20γZ, pH30γZ et pH45γZ et digérés avec la protéinase K en présence d'un tampon isotonique (Sucr., lignes 1, 3 et 5) ou un tampon hypotonique (H₂O, lignes 2, 4 et 6). Les marqueurs de poids moléculaire (en kilodaltons) sont indiqués sur la gauche.
• Figure 8 - Co-localisation des protéines P20γZ avec les α- et les γ-zéines dans les corps protéiques de l'endosperme du maïs. Une analyse en immunocytochimie a été réalisée sur des sections ultrafines en utilisant des anticorps αPL (marqués avec des particules d'or de diamètre 15 nm) et des anticorps αZ et αG2 (marqués avec des particules d'or de 5 nm). (A) Corps protéiques de l'endosperme transformé avec pP20γZ, immunomarqués avec l'anticorps αPL. (B) Immunolocalisation de P20_γZ (marqué avec des particules d'or de 15 nm) et de l'α-zéine (marquée avec les particules d'or de 5 nm) dans les corps protéiques isolés à partir des endospermes transformés avec pP20γZ. (C) et (D) Immunolocalisation de P20γZ (marqué avec les particules d'or de 15 nm) et des γ-zéines (marquées avec les particules d'or de 5 nm) dans des corps protéiques isolés à partir des endospermes transformés avec pP20γZ. Les flèches indiquent les sections tangentielles des corps protéiques.
• Figure 9 - Séquence codante de l'ADNc la γ-zéine de maïs et la séquence d'acides aminés correspondante.
• Figure 10 - Séquence codante de l'ADNc de la H45γZ zéine de maïs et la séquence d'acides aminés correspondante.
  La séquence riche en lysine (28 acides aminés) est introduite entre les résidus d'acides aminés 92 et 119 de la séquence représentée à la Figure 10.
• Figure 11 - Séquence codante de l'ADNc de la P20γZ zéine de maïs et la séquence d'acides aminés correspondante.
  La séquence riche en lysine (14 acides aminés) est introduite entre les résidus d'acides aminés 92 et 119 de la séquence représentée à la Figure 11.
• Figure 12 - Cartes de restriction des plasmides pBin 19P20γZ et pBin 19H30γZ.
• Figure 13 - Endospermes des plants de maïs transgéniques accumulant de la γ-zéine enrichie en lysine
  • A et B: SDS-page et Immunoblot utilisant l'antisérum αPL
    • A) 10 µg de protéine par piste (transformants avec le construit H45γZ)
      • piste C: extrait protéique des endospermes d'hybrides B73×A188 (contrôle)
      • piste 1: A1
      • piste 2: B1
      • piste 3: B2
      • piste 4: C1
      • piste 5: D1
      • piste 6: D2
    • B) 1 µg de protéine par piste (transformants avec le construit P20γZ)
      • piste C: contrôle
      • piste 1: A1
      • piste 2: A2
      • piste 3: B1
      • piste 4: C1
      • piste 5: D1
      • piste 6: E1
    • C) SDS-page et coloration à l'argent (3 transformants P20γZ et 3 transformants H45γZ)
      
      
• Figure 14 - Contenu en γ-zéine enrichie en lysine par grain (transformants 45γZ B1 et C1)
  • A: coloration à l'argent ; 10 µg de protéine par piste
  • B: immunoblot utilisant l'antisérum αPL ; 1 µg de protéine par piste
    • Pistes 1 à 5: extraits protéiques de différents endospermes du transformant 45γZ B1.
    • Pistes 6 à 10: extraits protéiques de différents endospermes du transformant 45γZ C1.
• Figure 15:
  • A) Contenu en γ-zéine enrichie en lysine de 10 grains (transformant 45γZ C1) utilisant l'antiserum αPL 1 µg de protéine par piste
    • Pistes 1 à 10: extrait d'endospermes de 10 descendants
  • B) Immunoblot des extraits protéiques des endospermes 1 à 5 présents en A) et marqués avec l'antisérum αG2; 2 µg de protéine par piste.

Other features and advantages of the invention appear in the examples and in the figures which follow.

• Figure 1
  • Restriction map of plasmid pP20γZ
• Figure 2
  • Restriction map of plasmid pH45γZ
• Figure 3
  • Schematic representation of the proteins encoded by the modified and unmodified γ-zein genes: wild type γ-zein (γZ), lysine-rich γ-zeins (P20γZ, H30γZ, H45γZ and N13γZ) resulting from the insertion of the oligonucleotides coding for lysine-rich sequences. The amino acid sequence of the inserted polypeptides is indicated using the one-letter code of the amino acid representation. The following abbreviations are used:
    • Term: terminal;
    • ProX DOMAIN: proline-Xaa linker domain.
• Figure 4 - In-vitro analysis of γ-zeins rich in lysine. (A) in vitro translation and translocation of transcripts corresponding to modified lysine-rich γ-zeins; Lines 1, 5, 9 and 13: complete translation products; Lanes 2, 6, 10 and 14: complete translation products after translocation in canine microsomes (CM); lanes 3, 7, 11 and 15: translocation products resistant to the action of proteinase K (PK); lanes 4, 8, 12 and 16: all of the translation products after treatment with proteinase K in the presence of 0.5% Nonidet P40 (NP40). (B) Immunoprecipitation of the in vitro translation products corresponding to the modified γ-zein and γ-zein proteins rich in lysine, using the anti-serum αPL. Lane 1: γ-zein; line 2: P20γZ; line 3: H30γZ; line 4: H45γZ and line 5: N13γZ. (C) Same legend as for (B) but using αG2 antiserum. Molecular weight markers (in kilodaltons) are shown on the left.
• Figure 5 - Tissue-specific activity of the γ-zein promoter. Endosperms of maize, embryos and maize leaves were co-processed by bombarding particles using the constructions shown in the figure (in the right-hand part). The relative activities of luciferase (LUC, columns in gray) and β-glucuronidase (GUS, hatched columns) were expressed as a multiplier of the values obtained with bare projectiles ± the standard deviation of the different ratios.
• Figure 6 - Expression of lysine-rich γ-zeins in the sub-aleuronic endosperm of cells. (A) Immunoblot with αPL antiserum, proteins extracted from endosperms transformed with pN13γZ (lane 2), pH45γZ (lane 3), pH30γZ (lane 4) and pP20γZ (lane 5). The control (line 1) corresponds to untransformed endosperms. Molecular weight markers (in kilodaltons) are shown on the left. (B) expression of H45γZ and N13γZ transcripts in transiently transformed endosperms. CDNAs obtained from tissues transformed with pH45γZ (lane 2), pN13γZ (lane 3) and the control (lane 1) were amplified by PCR and analyzed using a synthetic oligonucleotide encoding a lysine-rich sequence as a probe .
• Figure 7 - Accumulation of γ-zeins rich in lysine in the protein bodies of the endosperm. (A) Immunoblot analysis using αPL antiserum, protein bodies isolated from endosperms transformed with pP20γZ (lane 1), pH30γZ (lane 2), pH45γZ (lane 3), pN13γZ (lane 4) and no DNA ( line 5). (B) Immunoblot analysis using αPL antiserum, protein bodies isolated from endosperms transformed with pP20γZ, pH30γZ and pH45γZ and digested with proteinase K in the presence of an isotonic buffer (Sucr., Lines 1, 3 and 5) or a hypotonic buffer (H ₂ O, lines 2, 4 and 6). Molecular weight markers (in kilodaltons) are shown on the left.
• Figure 8 - Co-localization of P20γZ proteins with α- and γ-zeins in the protein bodies of corn endosperm. Immunocytochemistry analysis was performed on ultrafine sections using αPL antibodies (labeled with 15 nm diameter gold particles) and αZ and αG2 antibodies (labeled with 5 nm gold particles). (A) Protein bodies of the endosperm transformed with pP20γZ, immunolabeled with the αPL antibody. (B) Immunolocalization of P20 _γ Z (labeled with 15 nm gold particles) and α-zein (labeled with 5 nm gold particles) in the protein bodies isolated from the endosperms transformed with pP20γZ. (C) and (D) Immunolocalization of P20γZ (labeled with 15 nm gold particles) and γ-zeins (labeled with 5 nm gold particles) in protein bodies isolated from endosperms transformed with pP20γZ. The arrows indicate the tangential sections of the protein bodies.
• Figure 9 - Coding sequence of the maize γ-zein cDNA and the corresponding amino acid sequence.
• Figure 10 - Coding sequence of the H45γZ zein cDNA and the corresponding amino acid sequence.
  The lysine-rich sequence (28 amino acids) is introduced between amino acid residues 92 and 119 of the sequence shown in Figure 10.
• Figure 11 - Coding sequence of the p20γZ zein cDNA and the corresponding amino acid sequence.
  The lysine-rich sequence (14 amino acids) is introduced between amino acid residues 92 and 119 of the sequence shown in Figure 11.
• Figure 12 - Restriction maps of plasmids pBin 19P20γZ and pBin 19H30γZ.
• Figure 13 - Endosperms of transgenic corn plants accumulating γ-zein enriched in lysine
  • A and B: SDS-page and Immunoblot using αPL antiserum
    • A) 10 μg of protein per lane (transformants with H45γZ construct)
      • lane C: protein extract of hybrid endosperms B73 × A188 (control)
      • lane 1: A1
      • track 2: B1
      • lane 3: B2
      • lane 4: C1
      • lane 5: D1
      • lane 6: D2
    • B) 1 μg of protein per lane (transformants with the construct P20γZ)
      • track C: control
      • lane 1: A1
      • Track 2: A2
      • lane 3: B1
      • lane 4: C1
      • lane 5: D1
      • Track 6: E1
    • C) SDS-page and silver staining (3 transformants P20γZ and 3 transformants H45γZ)
      
      
• Figure 14 - Content in γ-zein enriched with lysine per grain (transformants 45γZ B1 and C1)
  • A: silver staining; 10 μg of protein per track
  • B: immunoblot using αPL antiserum; 1 μg of protein per track
    • Lanes 1 to 5: Protein extracts of different endosperms of the transformant 45γZ B1.
    • Lanes 6 to 10: Protein extracts of different endosperms of the transformant 45γZ C1.
• Figure 15:
  • A) Content in γ-zein enriched in lysine of 10 grains (transforming 45γZ C1) using antiserum αPL 1 μg of protein per lane
    • Tracks 1 to 10: endosperm extract of 10 offspring
  • B) Immunoblot protein extracts of endosperms 1 to 5 present in A) and labeled with αG2 antiserum; 2 μg of protein per lane

EXAMPLES A) Preparation of modified γ-zeins enriched in lysine and expression of these modified proteins followed by accumulation in the protein bodies of the cells of the corn endosperm

Γ-zein is a corn-rich reserve protein with a molecular weight of 28 kD, rich in sulfur, which is accumulated in endosperm cells with α- and β-zeins in protein bodies derived from the reticulum. granular endoplasmic (ER) (Ludevid et al., 1984. Plant Mol Biol 3, 227-234, Lending et al., 1984. Plant Cell 1, 1011-1023). The deduced amino acid sequence of the nucleotide sequence of the cDNA (Prat et al., 1985, Nucl Acids Res.13, 1493-1504) and genomic clones (Boronat et al., 1986, Plant Sci. , 95-102) shows that γ-zein has no homology with α-zein polypeptides. Although γ-zein is encoded by 1 to 2 genes per haploid genome (Boronat et al., 1986, Plant Sci., 47, 95-102) it accounts for 10-15% of the total endosperm protein of maize. The expression of the γ-zein gene in heterologous systems such as Xenopus oocytes (Torrent et al., 1994, Planta 192, 512-518) and in Arabidopsis thaliana (Geli et al., 1994, Plant Cell 6 , 1911-1922), indicates that γ-zein polypeptides remain stable and are capable as such of forming endoplasmic reticulum derived protein bodies within the cells. In addition, deletion analyzes of different structural domains of γ-zein showed that the N-terminal sequence including the proline-rich repeat domain was responsible for the retention of γ-zein in the endoplasmic reticulum and the cysteine-rich C-terminal domain was responsible for the formation of the protein bodies. The Pro-X domain did not appear to be related to the stability of the protein nor to its targeted localization (Geli et al., 1994, Plant Cell 6, 1911-1922).

Material and methods Plant material

After surface sterilization (1) of stage 17 DAP ("days after pollination") seeds of the selection corn W64A were dissected by hand and the pericarp and aleurone layer was separated from the endosperms. Tangential sections have been made to expose a large part of the sub-aleuronic surface. If necessary, embryos were isolated and 7 days old plant leaves were dissected to remove the epidermal tissue. After dissection, the samples were placed in petri dishes on filter papers moistened with MS medium (Murashige and Skoog, 1962, Physiol Plant 15, 473-497).

Plasmid constructs

A first set of plasmids, pKSG2, pHbP2, pPbP4 and pNaN1 was obtained to allow the introduction of restriction sites into the gene encoding γ-zein. pKSG2 and pHbP2 were constructed as described in Torrent M. et al. (Planta (1994) 192: 512-518). Plasmid pKSG2 contains the coding sequence of γ-zein.

The plasmid pHbP2 is obtained from pKSG2 and contains a coding sequence for a mutated γ-zein in which the Pro-X domain of the protein has been deleted.

Plasmid pPbP4 was obtained following two cloning steps: (i) the 350 bp SalI-Pvull restriction fragment of pKSG2 was cloned into a restricted plasmid (pBSKS, Stratagene, La Jolla, California USA) SalI and EcoRV (pKSC4) and (ii) the 600 bp Pvull-XbaI restriction fragment of pKSG2 was cloned into the Smal-XbaI restriction sites of pKSC4. The new pPbP4 construct contains a useful EcoRI restriction site just before the P-X domain of the γ-zein coding sequence.

The plasmid pNaN1 was also obtained using two cloning steps: (i) the 250 bp Nael-XbaI fragment of pKSG2 was cloned into the plasmid pBSKS restricted with EcoRV-XbaI (pKSC8) and (ii) the A 700 bp (blunt ended) Nael-HindIII restriction fragment of pKSG2 was cloned into the HindIII restriction site of pKSC8. The new construct, pNaN1, contains Clal and HindIII restriction sites at a position 15 nucleotides before the stop codon of γ-zein.

Two synthetic oligonucleotides corresponding to the following sequences: SEQ ID No. 1:
5'CGATGAATTCAAACCAAAGCCAAAGCCGAAGCCAAAAGAATTCA3 ', and its reverse sequence called SEQ ID No. 2, the sequence of which is as follows:
5'AGCTTGAATTCTTTTGGCTTCGGCTTTGGCTTTGGTTTGAATTCAT3 'coding for lysine-rich sequences designated K (PK) ₄ , were hybridized, digested with EcoRI and cloned into an EcoRI site of pHbP2 and pPbP4. Three clones were selected: pPo2 and pHo3 containing the coding sequence for K (PK) ₄ and pHo4 comprising the truncated form of the coding sequence for the γ-zein containing a 2K (PK) ₄ tandem (in the form of a K (PK) ₄ EF K (PK) ₄ sequence) of the lysine-rich coding sequence. The same hybridized oligonucleotides were digested with ClaI-HindIII enzymes and cloned into the restricted pNaN1 plasmid using the same enzymes. The selected clone, pNo1, contained the coding sequence for the lysine-rich K (PK) ₄ sequence at the N-terminus of the corresponding modified γ-zein.

For the transient transformation of the endosperm, the sequences coding for the modified γ-zein of pPo2 and pHo3 were inserted in the form of HincII-NheI fragments in SmaI-XbaI sites of pDH51 (Pietrzah et al., 1986, Nucl Acid Res 14, 5857-5868) containing the 35S promoter of Cauliflower Mosaic Virus (CaMV). The construction pP20γZ obtained by the insertion described above of the Hincli-Nhel fragments in the plasmid pDH51 contains the coding sequence of the γ-zein enriched in lysine (FIG. 8) as well as the signals of the 35S sequence of the CaMV virus for the formation of the 3 'end and polyadenilation. The P20γZ chimeric coding sequence was constructed from the coding region of the γ-zein contained in the plasmid pKSG2 after various cloning steps. The 1.7 kb promoter of γ-zein (Reina et al 1990, Nucl Acids Res 18, 6426) was inserted into the blunt ends of a HindIII-Pvu I fragment in pHo4 and pNo1 restricted with Xho I and obtained with blunt ends. The constructions pH45γZ and pN13γZ were respectively obtained.

The new constructs, respectively called pP20γ-Z, pH30γZ, pH45γZ and pN13γZ were used in biolistic bombardment experiments.

To study the specificity of different promoters vis-à-vis plant tissues, two constructions. p1.7γZGUS and pCaMV35SLUC were used. p1.7γZGUS was obtained by insertion of the γ-zein promoter 1.7 kb (HindIII-PvuI) in a plasmid derived from pPuC18 containing the GUS gene and the 3 'polyadenylation NOS signals of pBI 101.1 (Jefferson et al., 1987, Embo., J. 6, 3901-3907) . pCaMV35SLUC was obtained by insertion of the gene encoding luciferase (LUC) from pAHC18 (Bruce et al., 1989, PH 86, 9692-9696) into the polylinker of pDH51 (Pietrzak et al., 1986, Nucl Acids Res. 18, 6426).

In-vitro analysis

Plasmids derived from pBSKS containing the coding sequences of γ-zein (pKSG2) and lysine-rich γ-zein (pPo2, pHo3, pHo4 and pNo1) were transcribed in vitro according to the standard protocols (Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Ed., Cold Spring Harbor, New York). In vitro translation and translocation of the synthetic transcripts were performed according to the technique of Torrent et al., (1994, Panta 192, 512-518), except for the canine microsomes (CM) used which came from Promega ( Madison, Wis., USA). Immunoprecipitation of the translated products was carried out essentially according to the method of Borgese and Gaetani (1980) using anti-gamma-zein-G2 rabbit serum (Ludevid et al., 1985, Plant Sci 41, 41-48). ) and αPL antiserum. αPL is a rabbit polyclonal antiserum obtained against the synthetic peptide EFK (PK) ₈ EF. This peptide was synthesized using the solid phase synthesis technique described by Celma et al., 1992.

Microprojectile bombardment

Plasmid DNA was absorbed onto gold particles (1.0 μm, Bio-Rad, Lab., Richmond, CA, USA) according to a protocol described by Kikkert (Plant Cell, 33: 221-226, 1993). All targets were bombarded twice, using the BioRad Biolistic PDS-1000 / He device. The targets were positioned 8 cm behind a screen stopping the macroporteurs, which were positioned 1 cm below the rupture disc 900 PSI. After bombardment, the samples were incubated for 24 hours at 26 ° C in the dark. Controls consisted of targets bombarded with microprojectiles devoid of DNA.

Enzymatic tests

Tissues bombarded with plasmids p1.7γZGUS and pCaMV35SLUC were homogenized on ice in a buffer containing 25mM Tris, pH 7.8, 2mM DTT, 10% glycerol and 1% Triton X-100. After centrifugation at 12,000xg for 5 minutes, the supernatants were decanted and the total soluble protein in the extracts quantified using the Bradford test (Bio-Rad). GUS activity was assayed by fluorimetric analysis as described by Jefferson (1987) using 4-methyl-ombelliferyl-β-D-glucuronide (MUG) as the substrate. LUC activity was determined using the Luciferase Assay System Kit released by Promega, according to the manufacturer's instructions.

Extraction of reserve proteins and protein gel analysis

Endosperms transformed with pP20γZ, pH20γZ, pH45γZ and pN13γZ were reduced to flour state and α-zeins were extracted using three sets of solvents containing 70% ethanol. The residual meal was air-dried, and the total proteins were extracted with a buffer containing 0.25 M Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol for 1 hour at room temperature. Protein extracts were analyzed by SDS-PAGE and immunoblot as described by Ludevid et al., 1985. Nitrocellulose sheets were intubated with αPL antiserum (1: 500 dilution) and horseradish peroxidase conjugate. with a secondary antibody (ECL Western Blotting System, Amersham, Buckinghamshire, UK) was used for the detection of the protein.

Analysis of RNA expression

Total RNA was extracted according to the description of Logemas et al., 1987. The complementary DNA (cDNA) was prepared using reverse transcriptase and Gibco BRL oligo dT (Gaithersburg, MD, USA) according to manufacturer's instructions and this RNA was amplified by a PCR reaction. Oligonucleotide primers used for PCR were 20-mer sequences corresponding to the 5 'and 3' ends of the γ-zein structural gene. Standard protocols were used for the preparation of ³² P labeled probes, and for DNA gel analysis (Sambrook et al., Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Ed. , Cold Spring Harbor, New York) using a synthetic oligonucleotide encoding a lysine-rich sequence (see above) as a probe.

Isolation of protein bodies and protease treatment

These protocols have been described previously (Torrent et al., Planta, 180: 90-95, 1989).

Electron microscopy

The protein bodies of wild-type endosperms and endosperms transformed with pP20γZ were fixed with 2.5% paraformaldehyde in 20 mM phosphate buffer pH 7.2 for 1 hour at room temperature, and transformed according to Geli's description. al., 1994, Plant Cell 6. 1911-1922), however, using an α-PL antiserum and a colloid of gold and protein A having a diameter of 15 nm. For double labeling, ultra fine sections were first incubated with α-PL and gold colloid and protein A (15 nm in diameter) was used for antibody detection. After washing, sections were incubated with 0.15 mg / ml of protein A for 20 minutes to saturate the immunoglobulins and finally the grids were incubated with sera α-G2 or α-Z1 and the colloid gold / protein A ( diameter 5 nm) was used for the detection of the antibody. α-Z1 is a rabbit polyclonal antiserum directed against α-zein obtained according to the description of Ludevid et al., 1985. Plant Sci. 41, 41-48).

Results Construction of gamma-zeins rich in lysine

The inventors had demonstrated the importance of the proline-rich repeat domain and the cysteine-rich C-terminal domain for retention in the γ-zein endoplasmic reticulum and the formation of the protein bodies containing these proteins. proteins in Arabidopsis leaf cells (Geli et al., 1994, Plant Cell 6, 1911-1922). On the basis of these preliminary results, the possibility of inserting lysine-rich sequences in different domains of γ-zein, so as to create a properly targeted modified γ-zein accumulated in Endosperm cells have been investigated to improve the nutritional qualities of corn.

The inventors have now constructed modified γ-zein genes by introducing synthetic oligonucleotides encoding lysine-rich sequences in different sites of the γ-zein coding sequence. Modified γ-zein constructs have been created to avoid placement of lysine-rich coding sequences in the domains consisting of the tandem repeat domain and the cysteine-rich domain. Modifications of the coding sequence of the γ-zein were carried out in the sequence corresponding to the Pro-X domain. In addition, to minimize impairment of protein folding, the lysine-rich (PK) _n sequences have been defined to mimic the sequence of the Pro-X domain. As seen in FIG. 3, in the P20γZ protein, a K (PK) ₄ sequence was introduced after the Pro-X region and in the H30γZ protein and in the H45γZ protein, amino acid sequences comprising K ( PK) ₄ and 2K (PK) ₄ respectively replace the Pro-X domain of γ-zein (γZ, Fig. 3). To explore whether the C-terminus was a permissive site for the introduction of lysine-rich sequences, an additional protein N13γZ, was created by inserting a sequence containing K (PK) ₄ five amino acids upstream from the C-terminus (Fig.3).

Activity of γ-zein promoter in processed corn endosperm

To determine whether lysine-rich γ-zeins could be expressed in the endosperm cells, an efficient promoter and a transformation system were first sought. A γ-zein promoter, containing an upstream sequence of 1.7 Kb (Reina et al., 1990, Nucleic Acid Research, Vol 18, p 6426) and the CaMV promoter containing 625 bp of the upstream sequence of CaMV mosaic virus 35S protein were tested. So far, no information was available on the functional analysis of gene fusions with the γ-zein promoter in transgenic monocotyledonous plants. To analyze the activity and tissue specificity of the γ-zein promoter, two chimeric genes were constructed (see Fig. 5). transient expression by biolistic bombardment (Klein et al., 1988 PNAS 85: 4305) was used as a maize transformation procedure for promoter analysis as well as for lysine-rich γ-zein expression experiments. . Endosperms of stage 17 DAP maize (17 days after polynisation) (pericarp and aleuronic layer cells were removed), embryos (17 DAP) and young leaves (10 days old) were bombarded with gold bullets coated with plasmid DNA containing both constructs. FIG. 5 shows the β-glucuronidase (GUS) and luciferase (LUC) activities present in the three tissues tested: the endosperm, the embryo and the leaf, compared to the control experiment. It should be noted that the results correspond to the average of at least 3 independent experiments performed. All GUS activity under the control of the γ-zein promoter was restricted to the endosperm, since no GUS expression was detected in the embryo and in the leaves. In addition, the bombarded endosperms were stained histochemically to determine the number of cell clusters expressing the GUS protein. The staining pattern corroborated the previous results, GUS was strongly expressed in the endosperms (the average number of endosperm-stained cell clusters for GUS was 150) and blue spots were not detected in the embryo and in the leaves . In contrast, the CaMV 35S promoter conferred LUC activity in all tissues tested (Fig. 5), but differences existed. between the relative activity of the enzyme in the leaves and embryos compared to the endosperm. These differences could be attributed to an intrinsic variability in the constitutive activity of the CaMV promoter among different maize tissues or to a low penetration of the DNA-coated particles into mesophyl cells containing a large vacuolar system. There is evidence in the state of the art that the CaMV promoter would usually have low activity in monocotyledonous plant cells. (Fromm et al., 1985, Proc Natl Acad Sci USA 82, 5824-5828); however, the inventors have shown strong activity of the CaMV promoter in endosperm cells. This led to the conclusion that the activity of the γ-zein promoter and the CaMV 35S promoter was very strong in corn endosperms and that both promoters could prove useful for controlling the expression of the protein in this endosperm. tissue.

To determine whether the mutant proteins encoded by the constructs were competent with the membrane translocation function, in vitro transcription-translation experiments were performed in the presence of pancreatic dog microsomes. The synthetic transcripts of each construct were translated and translocated across the microsome membranes tested for protection against proteinase K digestion. These results reported in Figure 4A indicate that the apparent molecular weights of polypeptides synthesized in-vitro reflect the mutations introduced (Figure 4A, lines 1, 5, 9 and 13). In the presence of microsomes and proteinase K, low molecular weight peptides were not observed, indicating that the complete polypeptide chains of the modified γ-zeins were transported across the microsomal membranes (Fig. 4A, lines 3, 7). , 11 and 15). By comparing the result of the translocation of the four modified γ-zeins, it was observed that the protein H45γZ (Fig. 4A, line 11), which contained the insertion of 10 lysine-like amino acids, underwent a lower translocation than the other proteins. It seems that the negatively charged residues could interfere in a certain proportion, on the efficiency of the translocation. The αG2 polyclonal antibody, directed against γ-zein (Ludevid et al., 1985, Plant Sci. 41, 41-48) can not be used to distinguish between the wild-type γ-zein and the modified γ-zeins, an αPL antibody directed against a synthetic peptide containing the lysine-rich amino acid sequence was prepared. It was then tested whether modified proteins synthesized in vitro were recognized by both αG2 and αPL antibodies. This experiment is illustrated in FIG. 4B and FIG. 4C in which the synthetic transcripts of γ-zein, P20γZ, H30γZ, H45γZ and N13γZ were translated in vitro and in which the translation products were immunoprecipitated with αPL (FIG. and with αG2 (Fig. 4C). These results indicate that lysine-rich γ-zeins were recognized by both antibodies (see Fig. 4B and C, lines 2-5) and that γ-zeine was only recognized by αG2 serum (Fig. 48 and C, line 1). Thus, the specificity of the αPL antibodies for the modified proteins made it possible to distinguish the lysine-rich γ-zein from the endogenous γ-zein when the modified genes were expressed in endosperm cells. Taken together these experiments showed that the presence of lysine-rich sequences did not interfere with the translocation function across the membrane or the immunological behavior of γ-zein.

Analysis of the expression γ-zeins enriched in lysine in corn endosperms

To explore whether modified lysine-rich γ-zein was expressed and accumulated in endosperm cells, stage 17 seeds DAP were bombarded with the DNA containing the protein coding sequences of the four constructs (Fig. 3) under the control of the CaMV promoter (P20γZ and H20γZ) and the γ-zein promoter (H45γZ and N13γZ). Constructs comprising antisense promoters or lacking promoters were used as controls. After 24 hours of transformation of the endosperms, the total proteins were extracted and the expression of the modified γ-zeins was tested by immunoblotting using the αPL antibody. FIG. 6A shows that the γ-zein chimeric genes containing the insertion (Pro-Lys) _n after the Pro-X domain (P20γZ) or the substitute (H45γZ and H30γZ) were strongly expressed and the translation products accumulated efficiently in the endosperm cells (Fig. 6A, lines 3, 4 and 5). For each line, the protein extracts corresponded to approximately 1/3 of a bombarded endosperm, which made it possible to estimate that the amount of modified proteins P20γZ, H30γZ and H45γZ per endosperm reached the nanogram level. In addition, no quantitative difference between the levels of expression of the chimeric genes under the control of the CaMV promoter and the γ-zein promoter was observed, confirming the results described above obtained with the GUS and LUC marker proteins. (Fig. 5). It was noted that the αPL antibody recognizes an approximately 30 kD protein present in the total protein extracts, even in the untransformed endosperms (see the weak band present in the four lines of Figure 6A). A sequential protein extraction procedure revealed that this protein was not a soluble protein reserve in an aqueous medium.

As shown in FIG. 6A (line 2), no trace of the N13γZ protein was detected, indicating that the corresponding chimeric gene was not expressed in the endosperm cells or that the N13γZ protein was degraded. Endosperm RNAs transformed with the DNAs encoding the H45γZ and N13γZ proteins as well as the non-transformed endosperm RNAs were analyzed. From the total RNAs, the cDNAs were prepared and amplified by PCR using specific primers. Fig. 6B shows the Southem blot analysis of three cDNA samples hybridized with an oligonucleotide coding for a K (Pro-Lys) ₄ sequence used as a probe. The results indicate that the N13γZ gene was correctly expressed (Figure 6B, line 3). The presence of bands in the H45γZ and N13γZ samples, but not in the untransformed endosperms, suggested that the N13γZ protein was degraded during the 24 hours of incubation. From these observations, the inventors concluded that the insertion site of the lysine-rich sequences was critical for the stability of the modified γ-zein.

Γ-zein enriched in lysine is accumulated in the protein body

Apart from the lysine content of the Pro-X sequences, the P20γZ, H30γZ and H45γZ proteins had common characteristics with the wild-type γ-zein: the signal peptide, the N-terminal tandem repeat domain and the C-terminal cysteine were related. It seemed important to determine whether these domains remained fully functional preserving the targeting and formation of protein bodies or whether the lysine-rich sequences created a special environment in which these properties could be disrupted. To test this, it was investigated whether the modified γ-zeins were able to accumulate in the protein bodies. Sub-cellular fractionation was performed with the transformed endosperms. The bombarded endosperm homogenates were loaded onto discontinuous sucrose gradients (20%, 50% and 70% sucrose) and all fractions collected were analyzed by immunoblotting. P20γZ, H30γZ and H45γZ sedimented on the fraction of the protein bodies and no significant amount of these proteins was detected either in the supernatant or in the microsomal fraction (Fig. 7A, lines 2, 2 and 3). Although in previously- in vitro experiments (Fig. 4A) it was found that the newly synthesized modified γ-zeines were translocated in canine microsomes, it was tested here whether the modified proteins expressed in vivo in cells of endosperm, were translocated in the endoplasmic reticulum membrane and remained within the protein bodies derived from the endoplasmic reticulum. For this reason, isolated protein bodies have been digested with proteinase K in isotonic buffers (containing 20% sucrose) or after osmotic shock in water (Fig. 78). Proteins protected against proteolytic degradation by enzymes can be membrane-bound and treatment with detergent or hypotonic solutions has resulted in protein digestion (Walter and Blobel, 1983, Method Enzymol 96, 84-93). A comparison of the band intensities, after digestion with proteinase K in media comprising sucrose or water, revealed that the proteins P20γZ, H30γZ and H45γZ were protected from digestion in isotonic buffers (lines 1, 3 and 5) but were partially digested in water (lines 2, 4 and 6).

The expression of the modified γ-zein genes in the cells of the endosperm sub-aleurone layer by biolistic bombardment made it possible to observe that lysine-rich γ-zeins were largely accumulated with the exception of the case. where the lysine-rich sequences were positioned 5 residues upstream of the C-terminal end of the γ-zein polypeptide. From this expression and from immunocytochemical studies on isolated protein bodies, the inventors demonstrated that γ-zeins rich in lysine are correctly accumulated in these organelles and are co-localized with the endogenous proteins γ-zein and α-zein.

Protein bodies isolated from P20γZ endosperms were examined by immunogold-like staining and electron microscopy. On ultrathin sections incubated with αP-L antibody (Fig. 8A), gold labeling was detected within the protein bodies, indicating that the lysine-rich P20γZ protein was accumulated inside. of these organelles. In sections incubated only with αP-L antibody, immunostaining occurred only on a few protein bodies (containing lysine-rich γ-zein), most of the isolated protein bodies were not immunostained with antibodies αP-L because they corresponded to untransformed endosperm cells. To determine whether the γ-zein rich in lysine was co-localized with the α-zein and γ-zeine polypeptides, a double labeling in immunoelectromicroscopy was carried out on isolated protein bodies using the αZ and αP-L antibodies ( Fig. 8B) and αG2 and αP-L (Fig. 8C and D). Figure 8B shows a micrograph of the cross-section of two protein bodies labeled with the αP-L antibody (15 nm gold particle) and with the αZ antibody (5 nm gold particle). The result of the immunostaining showed that the protein P20γZ is accumulated in the protein bodies and co-localized with α-zein (see the extent of labeling of α-zein on the entire surface of the protein body). In addition, tangential (Fig. 8B, see arrows) and transverse (Fig. 8D) sections of the protein bodies were incubated with the αP-L antibody (15 nm gold particle) and with the αG2 antibody ( 5 nm gold particle). In both cases, the P20γZ protein was co-localized with γ-zein polypeptides. It was noted that the tangential sections of the protein body (Fig. 8A, C, see arrows) were easily distinguished from the cross-sections of the protein body in that the former had a higher density of electrons and that the labeling of the γ-zein was extended over the entire section. In contrast, the cross sections had a lower density and the labeling of the γ-zein was localized on the membrane surrounding the protein body. In both cases, the localization of the labeling of the γ-zein rich in lysine followed that of the endogenous γ-zeine.

B) Preparation of genetically modified plants, expressing γ-zeins rich in lysine 1) Obtaining and using maize callus as a target for genetic transformation.

The genetic transformation of maize, regardless of the method used (electroporation, Agrobacterium, microfibers, particle gun) generally requires the use of fast-dividing undifferentiated cells that have retained the ability to regenerate whole plants. This type of cells composes the friable embryogenic callus (called type II) corn.

These calli are obtained from immature embryos of genotype Hi II or (A188 x B73) according to the method and the media described by Armstrong (Maize Handbook, 1994) M. Freeling, V. Walbot Eds, pp 665-671 ). The calluses thus obtained are multiplied and maintained by successive subcultures every fortnight on the initiation medium.

Plantlets are then regenerated from these calluses by modifying the hormonal and osmotic balance of the cells according to the method described by Vain et al. (Plant Cell Tissue and Organ Culture (1989 18: 143-151). These plants are then acclimatized in a greenhouse where they can be crossed or self-fertilized.

2) Use of the particle gun for the genetic transformation of maize.

The preceding paragraph describes the obtaining and the regeneration of the cell lines necessary for the transformation; a genetic transformation method leading to the stable integration of the modified genes into the genome of the plant is described here. This method relies on the use of a particle gun identical to that described by J. Finer (Plant Cell Report (1992) 11: 323-328); the target cells are fragments of the calluses described in paragraph 1. These fragments with a surface area of 10 to 20 mm ² were placed, 4 hours before bombardment, at the rate of 16 fragments per box in the center of a petri dish containing a culture medium identical to the initiation medium, supplemented with 0.2 M mannitol + 0.2 M sorbitol. The plasmids carrying the genes to be introduced are purified on a Qiagen® column following the manufacturer's instructions. They are then precipitated on tungsten particles (M10) following the protocol described by Klein (Nature (1987) 327: 70-73). The particles thus coated are projected towards the target cells using the gun and according to the protocol described by J. Finer (Plant Cell Report (1992) 11: 323-328).

The boxes of calluses thus bombarded are then sealed with Scellofrais® and then grown in the dark at 27 ° C. The first subculture takes place 24h after, then every fortnight for 3 months on identical medium to the initiation medium supplemented with a selective agent whose nature and concentration may vary depending on the gene used (see paragraph 3). The selective agents that can be used generally consist of compounds certain herbicides (Basta®, Round up®) or certain antibiotics (Hygromycin, Kanamycin ...).

After 3 months or sometimes earlier, calluses whose growth is not inhibited by the selective agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies are obtained of the selection gene. The frequency of obtaining such calli is about 0.8 cal per box bombarded.

These calli are identified, individualized, amplified and then cultivated so as to regenerate seedlings (see paragraph 1). In order to avoid any interference with untransformed cells all these operations are carried out on culture media containing the selective agent.

Plants thus regenerated are acclimated and then grown in a greenhouse where they can be crossed or self-fertilized.

3) Use of the Bar gene for the production of genetically modified maize plants having incorporated and expressing the H45γZ gene

The Streptomyces hygroscopicus Bar gene encodes a phosphinotricin acyl transferase (PAT) that inactivates the phosphinotricin-active molecule of Basta® herbicide by acetylation. The cells carrying this gene are thus made resistant to this herbicide and can be selected via it. For cereal transformation, the coding sequence of the Bar gene is under the control of a regulatory region allowing strong and constitutive expression in plant cells. Such a region is advantageously constituted by the promoter and the first intron of the rice actin gene as described by Mc Elroy (Mol Gen. Genet (1991) 231: 150-160).

This chimeric gene is cloned on a plasmid allowing its amplification by Escherichia coli. This plasmid (pDM 302 Cao (Plant Cell Report (1992) 11: 586-591) after amplification then column purification. Qiagen® can be used in genetic transformation of corn using, for example, the method described in the preceding example. In this case, the culture media intended for the selection of the transformed cells are supplemented with 2 mg / l of phosphinotricin.

For the introduction of the H45γZ gene, a so-called cotransformation technique is advantageously used: the selection genes (Bar) and of interest (H45γZ) are carried by independent plasmids. In the case of the use of the particle gun, the plasmids are coprecipitated on the tungsten particles, the total amount of DNA precipitated on the particles remaining identical to that in the standard protocol (5 μg of DNA for 2.5 mg of particles) each plasmid representing about half the total weight of DNA used.

Experience shows that with this method, the cointegration of plasmids in plant cells is the most frequent event (of the order of 90%) that is to say that practically every plant having integrated the gene Bar and selected through it will also carry the H45γZ gene. The level of coexpression (percentage of selected plants expressing the H45γZ gene) is usually of the order of 70%.

The genes thus introduced are generally related to the genetic sense, the H45γZ gene can thus advantageously be followed in the progeny through the resistance to the herbicide which is closely associated.

The amount of modified protein is determined by the methods described in Example A, in particular by immunoblotting on protein extracts of immature or mature corn kernels, pooled on BASTA-resistant plants.

4) Example explaining the step of introducing transgenes and in particular the gene coding for H45γZ, for modifying the opaque phenotype 2 of maize

Improvement of opaque maize 2 by introgression of the γ-zein gene enriched in lysine.

The transformed plants described in the previous example, which exhibit both Basta resistance and the expression of a lysine-enriched γ-zein are used. Their pollen is used to fertilize opaque corn plants 2 of the W64Ao2 line which contains only one γ-zein gene. This line was obtained from the Maize Stock Center. The plants of these F1 progenies are selected for their resistance to Basta and then self-fertilized. F2 seeds thus produced are analyzed for the opaque phenotype on a light table and the opaque grains or vitreous grains are sown and evaluated for Basta resistance. In the case where all the opaque grains are sensitive to Basta, the demonstration is made that the introduction of the γ-zein gene enriched in lysine in the plant considered, complements the opaque phenotype 2.

In a second step, in these resistant Basta plants, individuals of o2 / o2 genotype and having only one γ-zein gene on chromosome 7 are selected using the molecular probes coding for the Opaque 2 gene and for γ-zein. The latter revealing polymorphic restriction fragments and only individuals with the pattern typical of the W64o2 line are retained (Lopes M.A. et al., 1995, Mol Gen. Genet 19: 247, 603-613).

These individuals have a lysine content that is on average equivalent to that of corn o2, or even higher. From these individuals, any introgression into ELITE varieties of 'high lysine "is observed via the determination of BASTA resistance and the presence of the o2 allele detected by RFLP.

5) Expression of γ-zeins enriched in lysine in Arabidopsis thaliana.

To obtain a stable transformation, the P20γZ and pH30γZ plasmid constructs cloned into the Bluescript KS (-) plasmid were inserted as HincII / XbaI fragments into the binary vector pBin19 (Bevan, M. Nucl., Acids Res., 12: 8711). 8721 (1984)), containing the cauliflower mosaic virus (CaMV) 35S promoter and the 3 'end and polyadenylation signals of the octopine synthetase (ocs) gene. The new plasmids were named p19P20γZ and p19H30γZ (Figure 12).

The binary vectors containing the sequences coding for the P20γZ and H30γZ proteins (p19P20γZ and p19H30γZ) were transferred into the Agrobacterium tumefaciens strain LBA4404 . Arabidopsis ecotype RLD plants were transformed according to the method described by Valvekens, D., Van Montagu, M. and Van Lijsebettens, M. ((1988) Proc Natl.Acad.Sci.USA 85: 5536-5540). For each construct, 10 transgenic plants were screened by immunoblot analysis using an antiserum obtained against γ-zein (αG2, Ludevid et al., 1985). The plants containing the highest levels (corresponding to about 0.1% of the total amount of proteins present in Arabidopsis leaves) of transgenic products in the F1 generation were chosen to obtain the F2 generation. These plants have also been selected for the expression of the desired protein.

Whole transgenic plants selected in medium containing kanamycin were homogenized in liquid nitrogen. Transgenic proteins were extracted selectively with a solution containing ethanol / 0.125 N hydrochloric acid HCl in a ratio of 3: 1 (v / v), with 5% mercaptoethanol and protease inhibitors. Proteins extracted with this solution were precipitated in 5 volumes of acetone and analyzed by SDS-PAGE and immunoblotting. Protein extracts from non-transgenic plants were used as controls. Proteins resulting from the insertion of K (PK) ₄ sequences into γ-zein were correctly expressed in Arabidopsis thaliana plants using the constitutive 35 S promoter of CaMV. On immunoblots, the αG2 and αPL antibodies recognized electrophoresis bands corresponding to the P20γZ and H30γZ proteins. These bands migrated with apparent molecular weights consistent with those previously observed in the in vitro translation / translocation experiments (30 kD and 26 kD, respectively). As observed in Arabidopsis transgenic plants expressing γ-zein (Geli et al., Plant Cell 6: 1911-1922 (1994)), the P20γZ and H30γZ proteins migrated in the form of two electrophoresis bands, namely the bands corresponding to 36 and 30 kD for P20γZ and the bands corresponding to 32 and 26 kD for H30γZ. Upper bands may be products that have undergone post-translational modifications. Such a post-translational modification was not detected in endosperms of processed maize. This result suggests that the modification would appear when these proteins are expressed in a heterologous system such as Arabidopsis thaliana.

6) Expression of recombinant lysine-enriched γ-zeins in corn. Method:

After obtaining the transgenic plants, they are crossed with a non-transformed male line. As a result, 50% of the grains harvested in this case of unilateral insertion will be transgenic. In order to analyze the γ-zeins enriched in lysine in transgenic plants, the proteins are extracted from 6 grains per transformant.

Endosperms are dissected by removing embryos and pericarps from seeds. The endosperms are milled and 50mg of flour is used for the selective extraction. Previously α-zeines are extracted by 3 treatments with 70% ethanol. After centrifugation, the ethanol is evaporated under vacuum. The insoluble proteins in ethanol (mainly γ-zeins and γ-zeins enriched in lysine) are extracted from the pellet with a Laemli buffer containing 10% of mercaptoethanol (100 μl of buffer for 10 mg of flour). The total proteins are then analyzed by silver staining (Morrissey, J.H., 1981, Ann Biochem, vol 117, pp. 307-310). The γ-zeins and γ-zeines enriched in lysine are analyzed by immunoblotting using the antibodies αG2 (dilution 1/2000) and αPL respectively (dilution 1/500). Alkaline phosphatase conjugated anti-rabbit antibody is used as a secondary antibody in the immunoblot. The extracts are diluted to allow their loading on SDS-page according to the analytical method used.

Results: Accumulation of γ-zeins enriched in lysine in 20γZ and 45γZ transgenic maize plants;

Figure 13 shows the immunoblot of protein extracts revealed with αPL antiserum (A, B) and total proteins after silver staining (C). As can be seen in FIG. 13A, 6 20γZ transgenic plants were tested with αPL antiserum and lysine-enriched γ-zein is expressed in transgenic A1 and D2 plants (lanes 1 and 6). respectively). In plant C1 (lane 4), only traces of γ-zein enriched in lysine are observed. When the extracts of the 6 45γZ transgenic plants are loaded onto the gel and labeled with the αPL antiserum (FIG. 13B), a strong reaction is observed with the antibody for transformants B1 and C1 (lanes 3 and 4, respectively).
It should be noted that the reaction with the antibody in the extracts of the endosperms of the plants 45γZ B1 and C1 is stronger than in the extracts of the plants 20γZ A1 and D2. This result is confirmed after silver staining of the gels (FIG 13C) where the two types of γ-zein: endogenous and enriched in lysine, are colored. The γ-zein enriched in lysine has an apparent molecular weight of 30kDa and the endogenous γ-zein of 28 kDa. The expression of γ-zein enriched in lysine is lower in the plant 45γZ B1 than in C1 (lanes 2 and 3 respectively). In lanes 1 to 6 of FIG. 13C, an identical dilution of the proteins of the endosperm extracts of the 45γZ and 20γZ plants was deposited on the gel. At this dilution, the expression of γ-zein enriched in lysine is detected only in the endosperms of the plants 45γZ B1 and C1. However, when a larger extract (40 μg of protein per lane) of 20γZ protein is loaded on the gel, a light band (see arrow), corresponding to the γ-zein enriched in lysine is detected in the endosperms 20γZ A1 and D2. This result indicates that the 45γZ plants accumulate much more proteins according to the invention than the 20γZ plants. This is probably due to the different activities of the promoters. The 45γZ plants were transformed with a construct containing the γ-zein according to the invention under the control of the γ-zein promoter (1.7 kb) whereas the 20γZ plants were transformed with the same coding sequence but under the control of the 35S promoter of CaMV. Silver staining is a general technique for staining proteins, but the strong brown color is mostly observed in the presence of basic proteins. Like the α-zeines were extracted from the flour, as described above, they are absent in the SDS-page analysis of Fig 13C.

Segregation

Since there is segregation of 50% in all transgenic plants in the case of a single locus, a grain-by-grain analysis was performed to quantify the level of lysine-enriched γ-zein in each transformant. The analysis was done only with the 45γZ transformants that had the highest expression level. Figure 14 shows silver staining (A) and immunoblot with αPL (B) of 5 endosperms different from 45γZ B1 and C1. The weak electrophoretic band present on all the tracks (see A, lanes 1 to 10) corresponds to the endogenous γ-zein. As can be seen in Fig. 14A, 2 of the 5 endosperms accumulate γ-zein enriched in lysine (see lanes 3,4 and 6,7). As a result, about half of the seeds accumulate significant amounts of lysine-enriched protein. Taking into account the fact that identical amounts of proteins have been deposited on the gel, it is observed that the 45γZ C1 transformant has accumulated more γ-zein enriched in lysine than B1. In fact, this result is in agreement with what is observed during the silver staining of the mixed extracts of endosperm (FIG. 13C, lane 3). The evidence for the presence of γ-zein enriched in lysine in these endosperms is highlighted in Fig 14B. The immunoblot using the αPL antiserum shows that 2 endosperm extracts of 45γZ B1 (lanes 3 and 4) and 45γZ C1 (lanes 4 and 6) accumulate γ-zein enriched in lysine. To confirm the percentage of transgenic grains, 10 new 45γZ C1 transformant seeds were analyzed by immunoblotting and use of αPL antiserum (Fig. 15A). As expected, about half of the transgenic seeds are detected. An immunoreactive band was observed in the endosperm extracts (lanes 1,2,9 and 10).

Estimation of the amount of γ-zeins enriched in lysine in the endosperms of 45γ Z transformants ;

αG2 is a polyclonal antiserum that recognizes endogenous γ-zeins and γ-zeins enriched in lysine. The reactivity of this antiserum with endosperm extracts 45γZ C1 was used to quantify the amount of lysine-enriched γ-zeins according to the invention in the endosperms of the transformed plants.

• i) dans les endospermes transgéniques 45γZ C1, le ratio γ-zéine enrichie en lysine/γ-zéine endogène est de 7/3. Donc la quantité de protéine modifiée selon l'invention est au moins 2 fois plus importante que celle de la protéine endogène,
• ii) la quantité de γ-zéine endogène dans les endospermes non transgéniques (voir pistes 3, 4 et 5) était équivalente à celle de la γ-zéine enrichie en lysine dans les endospermes transgéniques (voir pistes 1 et 2).

Figure 15B shows the immunoblot of 5 protein extracts corresponding to 5 endosperms 45γZ C1 (lanes 1 to 5). As expected, only 2 endosperms showed a characteristic immunoreaction pattern of transgenic grains. The upper band of 30 kDa corresponds to the γ-zein enriched in lysine (arrows on lane 1 and 2) and the lower band to the endogenous γ-zeine. It can be noted that in endosperms of non-transgenic plants the upper band is absent (lanes 3, 4 and 5). Surprisingly, it appears that the endogenous γ-zein content is lower in transgenic plant extracts than in non-transgenic plants (see arrows on lanes 1 and 5).
At first glance, it has been observed that:

• i) in endogenous endosperm 45γZ C1, the γ-zein ratio enriched in endogenous lysine / γ-zein is 7/3. Therefore, the amount of protein modified according to the invention is at least 2 times greater than that of the endogenous protein,
• ii) the amount of endogenous γ-zein in non-transgenic endosperms (see lanes 3, 4 and 5) was equivalent to that of γ-zein enriched in lysine in transgenic endosperms (see lanes 1 and 2).

7) Expression of recombinant lysine-enriched γ-zeins in wheat.

As in Example 6), it is possible to show the presence of γ-zeins enriched in lysine according to the invention in wheat.
The transformation of wheat can in particular be carried out according to the method described in Weeks et al., 1993, Plant Physiol., Vol 102: 1077-1084 or according to the method described in EP709462.

SEQUENCE LIST

• (1) GENERAL INFORMATION:
  • (i) DEPOSITOR:
    • (A) NAME: BIOCEM
    • (B) STREET: Cezeaux University Campus - 24 avenue des Landais
    • (C) CITY: AUBIERE
    • (E) COUNTRY: FRANCE
    • (F) POSTAL CODE: 63170
  • (ii) TITLE OF THE INVENTION: PROTEINS OF RESERVE OF PLANTS ENRICHED WITH AMINO ACIDS, IN PARTICULAR GAMMA-ZEINE OF MAIZE ENRICHED WITH LYSINE. PLANTS EXPRESSING THESE PROTEINS.
  • (iii) NUMBER OF SEQUENCES: 11
  • (iv) COMPUTER-DEPENDABLE FORM:
    • (A) SUPPORT TYPE: Floppy disk
    • (B) COMPUTER: IBM PC compatible
    • (C) OPERATING SYSTEM: PC-DOS / MS-DOS
    • (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO)
  • (v) DATA OF THE CURRENT APPLICATION:
    • NUMBER OF THE APPLICATION: PCT / FR 97/00167
• (2) INFORMATION FOR SEQ ID NO: 1:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 44 base pairs
    • (B) TYPE: nucleotide
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) MOLECULE TYPE: Other nucleic acid
    • (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE"
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
    
• (2) INFORMATION FOR SEQ ID NO: 2:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 46 base pairs
    • (B) TYPE: nucleotide
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) MOLECULE TYPE: Other nucleic acid
    • (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE"
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
    
• (2) INFORMATION FOR SEQ ID NO: 3:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 17 amino acids
    • (B) TYPE: amino acid
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: protein
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
    
• (2) INFORMATION FOR SEQ ID NO: 4:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 28 amino acids
    • (B) TYPE: amino acid
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: protein
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
    
• (2) INFORMATION FOR SEQ ID NO: 5:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 20 amino acids
    • (B) TYPE: amino acid
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: protein
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
    
• (2) INFORMATION FOR SEQ ID NO: 6:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 672 base pairs
    • (B) TYPE: nucleotide
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: cDNA
  • (ix) CHARACTERISTIC:
    • (A) NAME / KEY: CDS
    • (B) LOCATION: 1..669
    • (D) OTHER INFORMATION: / note = "CODING SEQUENCE OF GAMMA-ZEINE cDNA AND CORRESPONDING AMINO ACID SEQUENCE"
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
    
    
• (2) INFORMATION FOR SEQ ID NO: 7:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 223 amino acids
    • (B) TYPE: amino acid
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: protein
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
    
    
• (2) INFORMATION FOR SEQ ID NO: 8:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 693 base pairs
    • (B) TYPE: nucleotide
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: cDNA
  • (ix) CHARACTERISTIC:
    • (A) NAME / KEY: CDS
    • (B) LOCATION: 1..690
    • (D) ADDITIONAL INFORMATION: / note = "CODING SEQUENCE OF H45GAMMA-Z ZHEINE CORN cDNA AND CORRESPONDING AMINO ACID SEQUENCE."
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
    
    
• (2) INFORMATION FOR SEQ ID NO: 9:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 230 amino acids
    • (B) TYPE: amino acid
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: protein
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
    
    
• (2) INFORMATION FOR SEQ ID NO: 10:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 723 base pairs
    • (B) TYPE: nucleotide
    • (C) NUMBER OF BRINS: simple
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: cDNA
  • (ix) CHARACTERISTIC:
    • (A) NAME / KEY: CDS
    • (B) LOCATION: 1..720
    • (D) OTHER INFORMATION: / note = "CODING SEQUENCE OF P20GAMMAZ ZEINE CORN cDNA AND CORRESPONDING AMINO ACID SEQUENCE."
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
    
    
• (2) INFORMATION FOR SEQ ID NO: 11:
  • (i) CHARACTERISTICS OF THE SEQUENCE:
    • (A) LENGTH: 240 amino acids
    • (B) TYPE: amino acid
    • (D) CONFIGURATION: linear
  • (ii) TYPE OF MOLECULE: protein
  • (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
    
    

Claims (36)

1. Recombinant nucleotide sequence comprising a series of nucleotides coding for a storage protein from the zein family, characterized in that it furthermore comprises an oligonucleotide selected from amongst:

   a) an oligonucleotide comprising at least one series coding for a polypeptide of the formula (P-K)n in which:

   - n is an integer of 2 or above,

   - P represents a proline amino acid residue,

   - K represents a lysine amino acid residue,

   the symbol "-" denotes a bond between the two amino acid residues, in particular a peptide-type bond, the n units (P-K) likewise being linked to each other by such bonds, for example peptide-type bonds,

   b) an oligonucleotide as described in a) in which n is an integer of 3 or above, preferably n is 4, 5, 6, 7, 8, 9, 10 or 15,

   c) an oligonucleotide as described in a) or b) in which the sequence of the n units (PK) is interrupted by one or more amino acid residues which differ from the residues P or K,

   d) an oligonucleotide as described in a), b) or c) which is completed at its 5' and/or 3' end by one or more codons,

   e) an oligonucleotide as described in a), b), c) or d) which is completed at its 5' and/or 3' end by a lysine residue at the N-terminal end of the polypeptide formed,

   f) an oligonucleotide as described in e) which codes for a polypeptide of the formula (P-K)n, the formula K(P-K)₄ or the formula 2K(P-K)₄;

   said oligonucleotide being inserted at a site of the nucleotide series which is chosen in such a way that

   - the expression of the nucleotide sequence in a particular plant cell allows a modified storage protein of the zein family to be obtained whose localization is identical or similar to the normal storage protein which would be expressed by the corresponding coding nucleotide series under the same conditions in the same cell, and/or

   - the modified storage protein of the zein family which is encoded by the recombinant nucleotide sequence is recognized immunologically by antibodies raised against the corresponding normal storage protein.
2. Recombinant nucleotide sequence according to Claim 1, characterized in that the oligonucleotide is inserted at the N- or C-terminal end of the nucleotide series.
3. Recombinant nucleotide sequence according to Claim 1, characterized in that the oligonucleotide is inserted at proline-rich tandem repeats of the nucleotide series.
4. Nucleotide sequence according to one of Claims 1 to 3, characterized in that the nucleotide series codes for maize γ-zein.
5. Nucleotide sequence according to Claim 4, characterized in that the nucleotide series which codes for maize γ-zein has the sequence shown in Figure 9.
6. Nucleotide sequence according to Claim 4 or 5, characterized in that the oligonucleotide is inserted instead of or following the pro-X domain or in the pro-X domain which is naturally present in maize γ-zein.
7. Recombinant nucleotide sequence, characterized in that it comprises a nucleotide sequence according to any of Claims 1 to 6 under the control of an expression promoter.
8. Recombinant nucleotide sequence according to Claim 7, characterized in that the promoter is a promoter which is specific for a particular cell tissue, for example a promoter which is specific for expression in the seeds and/or the leaves of plants.
9. Nucleotide sequence according to Claim 8, characterized in that the expression promoter is the promoter of maize γ-zein.
10. Nucleotide sequence according to Claim 7, characterized in that the expression promoter is the CaMV35S promoter.
11. Nucleotide sequence according to any of Claims 6 to 10, characterized in that it codes for one of the polypeptides P20yZ or H45yZ, which have the sequences shown in Figures 11 and 10, respectively.
12. Cloning and/or expression vector, characterized in that it comprises a nucleotide sequence according to any of Claims 1 to 11 at a site which is not essential for its replication.
13. Cloning and/or expression vector, characterized in that it is one of the plasmids pP20yZ (CNCMN I-1640) or pH45yZ (CNCMN I-1639), or in that it comprises an oligonucleotide coding for the polypeptide H30yZ, which is shown in Figure 3.
14. Polypeptide encoded by a sequence according to any of Claims 1 to 6.
15. Lysine-rich modified maize γ-zein, characterized in that it is encoded by the nucleotide sequence according to Claim 5.
16. Lysine-enriched modified maize γ-zein, characterized in that its amino acid sequence is modified by at least one polypeptide of the formula (P-K)n or the formula K-(P-K)n in which:

    - n is an integer of 2 or above,

    - P represents a proline amino acid residue,

    - K represents a lysine amino acid residue,

    - the symbol "-" denotes a bond between the two amino acid residues, in particular a peptide-type bond, the n units (P-K) being linked by bonds, in particular peptide-type bonds, said polypeptide which has the formula (P-K)n or K-(P-K)n being inserted in the normal maize γ-zein amino acid sequence in such a way that

    - when the lysine-rich modified γ-zein is produced in a host cell, in particular a plant cell, its localization is identical or similar to normal maize γ-zein which would be produced under the same conditions in the same host cell, and/or

    - the modified maize γ-zein is recognized by antibodies directed against normal maize γ-zein.
17. Lysine-enriched modified maize γ-zein according to Claim 16, characterized in that said polypeptide is substituted for a proline-rich tandem repeat sequence which is naturally present in normal maize γ-zein.
18. Lysine-enriched modified maize γ-zein according to Claim 16, characterized in that said polypeptide is inserted with deletion of one or more amino acids of a proline-rich tandem repeat sequence of normal γ-zein.
19. Lysine-enriched modified maize γ-zein according to Claim 16, characterized in that said polypeptide is added into a proline-rich tandem repeat sequence of normal γ-zein.
20. Lysine-enriched modified maize γ-zein according to Claim 16, characterized in that said polypeptide is inserted in the N- or C-terminal part of the amino acid sequence of normal maize γ-zein.
21. Modified maize γ-zein according to Claim 14, characterized in that it is the protein P20yZ, Figure 11, or the protein H30yZ, Figure 3, or the protein H45yZ, Figure 10.
22. Recombinant host cell, characterized in that it comprises a nucleotide sequence according to any of Claims 1 to 11.
23. Host cell according to Claim 22, characterized in that it is a bacterium, for example E. coli or Agrobacterium tumefaciens.
24. Host cell according to Claim 22, characterized in that it is a plant cell.
25. Host cell according to Claim 24, characterized in that it is a plant seed cell.
26. Host cell according to Claim 25, characterized in that it is an endosperm cell or maize seed.
27. Host cell according to Claim 26, characterized in that it contains a nucleotide sequence according to Claim 5 stably integrated into its genome.
28. Host cell according to Claim 26, characterized in that it produces a lysine-rich modified maize γ-zein according to one of Claims 16 to 21.
29. Host cell according to Claim 24, characterized in that it is a soya, sunflower, tobacco, wheat, oat, lucerne, rice, oil seed rape, Arabidopsis or maize cell.
30. Seed producing a polypeptide according to any of Claims 16 to 21.
31. Plant producing a polypeptide according to any of Claims 16 to 21.
32. Plant according to Claim 31, characterized in that it is maize.
33. Seed expressing a modified polypeptide and obtained from plants according to Claim 31 or 32.
34. Use of an oligonucleotide selected from amongst:

    a) an oligonucleotide comprising at least one series coding for a polypeptide of the formula (P-K)n in which:

    - n is an integer of 2 or above,

    - P represents a proline amino acid residue,

    - K represents a lysine amino acid residue,

    the symbol "-" denotes a bond between the two amino acid residues, in particular a peptide-type bond, the n units (P-K) likewise being linked to each other by such bonds, for example peptide-type bonds,

    b) an oligonucleotide as described in a) in which n is an integer of 3 or above, preferably n is 4, 5, 6, 7, 8, 9, 10 or 15,

    c) an oligonucleotide as described in a) or b) in which the sequence of the n units (PK) is interrupted by one or more amino acid residues which differ from the residues P or K,

    d) an oligonucleotide as described in a), b) or c) which is completed at its 5' and/or 3' end by one or more codons,

    e) an oligonucleotide as described in a), b), c) or d) which is completed at its 5' and/or 3' end by a lysine residue at the N-terminal end of the polypeptide formed,

    f) an oligonucleotide as described in e) which codes for a polypeptide of the formula (P-K)n, the formula K(P-K)₄ or the formula 2K(P-K)₄ for obtaining a lysine-enriched storage protein of the zein family.
35. Method of producing plants or seed expressing a modified storage protein of the zein family, characterized in that it comprises the following steps:

    a) transformation of the plant cell with a nucleotide sequence according to any of Claims 1 to 11, or with a vector according to either of Claims 12 and 13 under conditions which allow the stable and functional expression of the modified storage protein encoded by the nucleotide sequence;

    b) regeneration of plants from the transformed plant cell of step a) for obtaining plants expressing the modified storage protein;

    c) if appropriate, obtaining seed from modified plants obtained in step b).
36. Method according to Claim 35, characterized in that the plant is maize and the storage protein is γ-zein.

Images